(T0930-04-19) Gemcitabine/Doxorubicin-Combination Polymer-Drug Conjugate Synthesized by SPAAC Click Chemistry: Characterization and Evaluation in OVCAR-3 Ovarian Cancer Cells

Purpose: Combination chemotherapy is preferred for the treatment of ovarian cancer (OC). Systemic toxicity, however, frequently limits the effectiveness of treatment. Polymer-drug conjugates (PDCs) containing synergistic combinations of chemotherapeutic drugs can be used to enhance therapeutic efficacy and minimize toxicity. This study aimed to develop a cathepsin B-sensitive PDC containing doxorubicin and gemcitabine for combination chemotherapy of OC.

Methods: Strain-promoted [3 + 2] azide-alkyne cycloaddition (SPAAC)-mediated step-growth polymerization method was used to prepare individual gemcitabine and doxorubicin PDCs and a gemcitabine/doxorubicin combination PDC. The cytotoxic effects of the PDCs were evaluated in vitro in OVCAR-3 OC cells using CellTiter-Glo assay and compared with the free drugs at equivalent concentrations.

Results: The gemcitabine/doxorubicin combination PDC had a high molecular weight (M_w >1000 kDa), high drug loading (36.6 %wt. gemcitabine; 7.0 %wt. doxorubicin), and narrow molecular weight distribution (M_w/M_n = 1.01). It demonstrated cathepsin B-catalyzed drug release at pH 5.0, and good stability at pH 7.4. Combination index analysis of free gemcitabine and free doxorubicin showed a concentration-dependent synergism (combination index < 1) in OVCAR-3 OC cells. Compared to individual gemcitabine PDC (IC₅₀ > 50 µg/mL) and doxorubicin PDC (IC₅₀ = 1.79 µg/mL), the combination PDC (IC₅₀ = 0.99 µg/mL) showed greater cytotoxicity against OVCAR-3 cells and being a conjugate, was less cytotoxic than the equivalent free drugs combination (IC₅₀ = 0.11 µg/mL). In addition, the cytotoxicity of the combination PDC where gemcitabine and doxorubicin were coupled to the same polymer backbone was greater than the cytotoxicity of the 50: 50 admixture of the individual PDCs (IC₅₀ = 3.87 ± 1.22 µg/mL).

Conclusion: The gemcitabine/doxorubicin combination PDC is promising for passively targeted combination chemotherapy of OC. In addition, the SPAAC-mediated step-growth polymerization method of synthesis of the PDCs was rapid and did not require the use of high temperature, initiator, or catalyst. In our opinion, this work is the first report of this method for the synthesis of PDCs.

Acknowledgements: This work was supported, in part, with federal funds from the National Cancer Institute, National Institutes of Health, under contract no. 75N91019D00024. The formulation described herein was accepted into the Assay Cascade Characterization Program of the Nanotechnology Characterization Laboratory (NCL) of the Frederick National Laboratory for Cancer Research. The NCL provides a free characterization service for cancer-related nanomedicine formulations, available to the public by application (https://www.cancer.gov/nano/research/ncl). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.